Cyclin-dependent protein kinases (“CDKs”) constitute a family of well-conserved enzymes that play multiple roles within the cell, such as cell cycle regulation and transcriptional control (Nasmyth, K., Science 1996, 274, 1643-1677; Morgan, D. O., Ann. Rev. Cell Dev. Biol. 1997, 13, 261-291).
Some members of the family, such as CDK1, 2, 3, 4, and 6 regulate the transition between different phases of the cell cycle, such as the progression from a quiescent stage in G1 (the gap between mitosis and the onset of DNA replication for a new round of cell division) to S (the period of active DNA synthesis), or the progression from G2 to M phase, in which active mitosis and cell division occur. Other members of this family of proteins, including CDK7, 8, and 9 regulate key points in the transcription cycle, whereas CDK5 plays a role in neuronal and secretory cell function.
CDK complexes are formed through association of a regulatory cyclin subunit (e.g., cyclin A, B1, B2, D1, D2, D3, and E) and a catalytic kinase subunit (e.g., cdc2 (CDK1), CDK2, CDK4, CDK5, and CDK6). As the name implies, the CDKs display an absolute dependence on the cyclin subunit in order to phosphorylate their target substrates, and different kinase/cyclin pairs function to regulate progression through specific portions of the cell cycle.
CDK9 in association with its cyclin partners (cyclin T1, T2a, T2b, or K) constitutes the catalytic component of the positive transcription elongation factor b (P-TEFb) protein complex that functions during the elongation phase of transcription by phosphorylating the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II. P-TEFb acts in concert with positive transcription factors as well as negative regulatory factors of RNA transcription, thus overcoming a block of transcriptional elongation (Liu, H., and Herrmann, C. H., J. Cell Physiol. 2005, 203, 251-260). Flavopiridol analogues that selectively inhibit P-TEFb were described recently (Ali, A. et al., Chembiochem. 2009, 10, 2072-2080).
It is known that cell-cycle dysregulation, which is one of the cardinal characteristics of neoplastic cells, is closely associated with genetic alteration and deregulation of CDKs and their regulators, suggesting that inhibitors of CDKs may be useful as therapeutics for proliferative diseases, such as cancer. Thus, small molecule inhibitors targeting CDKs have been the focus of extensive interest in cancer therapy (Dai, Y., and Grant, S., Current Opinion in Pharmacology, 2003, 3, 362-370; Zhang, J. et al., Nat. Rev. Cancer 2009, 9, 28-39). The ability of inhibiting cell cycle progression suggests a general role for small molecule inhibitors of CDKs as therapeutics for proliferative diseases, such as cancer. Recently, inhibition of CDK9/cyclin T function was also linked to prevention of HIV replication and the discovery of new CDK biology thus continues to open up new therapeutic indications for CDK inhibitors (Sausville, E. A., Trends Molec. Med. 2002, 8, S32-S37), such as, for example, viral infections (WO 02/100401). Further investigations on that field were published by Ali, A. et al. (Chembiochem. 2009, 10, 2072-2080). CDK inhibitors could conceivably also be used to treat other conditions such as immunological diseases and neurodegenerative diseases, amongst others.
More than 50 pharmacological CDK inhibitors have been described, some of which have potent antitumor activity (Dai, Y., and Grant, S., Current Opinion in Pharmacology, 2003, 3, 362-370). Recently, molecular markers for prediction of sensitivity of tumor cells towards CDK inhibitors were described (Eguchi, T. et al., Mol. Cancer. Ther. 2009, 8, 1460-1472). Contributions concerning the selectivity of protein kinase inhibitors was investigated and published by Bain, J. et al. (Biochem. J. 2007, 408, 297-315) and Karaman, M. W. et al. (Nat. Biotechnol. 2008, 26, 127-132). Furthermore, a comprehensive review about the known CDK inhibitors may be found in the literature (Huwe, A. et al., Angew. Chem. Int. Ed. Engl. 2003, 42, 2122-2138; Krug, M. and Hilgeroth, A. Mini. Rev. Med. Chem 2008, 8, 1312-1327). The use of 2-anilino-4-phenylpyrimidine derivatives as cyclin-dependent kinase inhibitors for the treatment of e.g. cancer has been reported in WO 2005/012262. Furthermore, 2-pyridinylamino-4-thiazolyl-pyrimidine derivatives for the treatment of cancer etc. have been described in WO 2005/012298. The use of 4,5-dihydro-thiazolo, oxazolo and imidazolo[4,5-h]quinazolin-8-ylamines as protein kinase inhibitors is known from WO 2005/005438. Furthermore, indolinone derivatives and indirubin derivatives, which are useful as cyclin-dependent kinase inhibitors have been disclosed in WO 02/081445 and WO 02/074742. Additionally, CDK inhibitors for various therapeutic applications have been described in WO2005/026129.
Known CDK inhibitors may be classified according to their ability to inhibit CDKs in general or according to their selectivity for a specific CDK. Flavopiridol, for example, acts as a “pan” CDK antagonist and is not particularly selective for a specific CDK (Dai, Y., and Grant, S., Current Opinion in Pharmacology, 2003, 3, 362-370). Purine-based CDK inhibitors, such as olomoucine, roscovitine, purvanolols and CGP74514A are known to exhibit a greater selectivity for CDKs 1, 2 and 5, but show no inhibitory activity against CDKs 4 and 6 (Dai, Y., and Grant, S., Current Opinion in Pharmacology, 2003, 3, 362-370). Furthermore, it has been demonstrated that purine-based CDK inhibitors such as roscovitine can exert anti-apoptotic effects in the nervous system (O'Hare, M. et al., Pharmacol Ther 2002, 93, 135-143) or prevent neuronal death in neurodegenerative diseases, such as Alzheimers's disease (Filgueira de Azevedo, W. Jr., Biochem Biophys Res Commun 2002, 297, 1154-1158; Knockaert, M. et al., Trends Pharmacol Sci 2002, 23, 417-425).
Given the tremendous potential of targeting CDKs for the therapy of conditions such as proliferative, immunological, infectious, cardiovascular and neurodegenerative diseases, the development of small molecules as selective inhibitors of particular CDKs constitutes a desirable goal.
Glycogen synthase kinase-3 (GSK3) was initially identified as an enzyme involved in the control of glycogen metabolism, in particular as a protein kinase that inactivates glycogen synthase. More recently, it has been shown to have key roles in regulating a diverse range of cellular functions by phosphorylating several target proteins, including transcription factors, metabolic enzymes, structural proteins and signaling proteins (Lee, J. et al., Diabetes Res Clin Pract. 2007, 77, Suppl 1:S49-57). Hence, GSK3 is regarded as a key enzyme regulating intracellular signal transduction pathways, thereby controlling cellular responses to extracellular and intracellular regulatory factors. Two isoforms of GSK3 have been described (GSK3-alpha and -beta) which are very similar to each other based on high sequence homology (86% overall and 97% in kinase domains) and biochemical characteristics. However, their physiological functions may not be fully redundant as genetic inactivation leads to different phenotypes in mice (Lee, J. et al., Diabetes Res Clin Pract. 2007, 77, Suppl 1:S49-57).
Based on the identification of cellular targets and genetic or pharmacologic modulation of GSK3 activity or expression, GSK3 appears to be involved in the molecular pathogenesis of several severe human diseases. For example, GSK3 inhibition has been suggested to exert therapeutic effects in human disorders including metabolic diseases, in particular diabetes (MacAulay, K. et al., Expert Opin Ther Targets. 2008, 12, 1265-1274; Rayasam, G. V. et al., Br J. Pharmacol. 2009, 156, 885-898; Lee, J. et al., Diabetes Res Clin Pract. 2007, 77, Suppl 1:S49-57), neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis and Huntington's disease (Hooper, C. et al., J. Neurochem. 2008, 104, 1433-1439; Martinez, A., Med Res Rev. 2008, 28, 773-796; Wada, A., Front Biosci. 2009, 14, 1558-1570; Huang, H. C. et al., Curr Drug Targets. 2006, 7, 1389-1397), parenchymal renal diseases (Obligado, S. H. et al., Kidney Int. 2008, 73, 684-690), neurologic and neuropsychiatric diseases including bipolar disorder (O'Brien, W. T. et al., Biochem Soc Trans. 2009, 37, (Pt 5) 1133-1138; Jope, R. S. et al., Curr Drug Targets. 2006, 7, 1421-1434), cardiovascular diseases including cardiac infarction and stroke (Juhaszova, M. et al., Circ Res. 2009, 104, 1240-1252), proliferative diseases including cancer (Luo, J. et al., Cancer Lett. 2009, 273, 194-200) and inflammatory disorders including multiple sclerosis, arthritis and colitis (Jope, R. S. et al., Neurochem Res. 2007, 32, 577-595).
Several GSK3 small molecule inhibitors have been identified, including amino-pyrimidines, thiadiazolidindiones, maleiimides, indirubins, paullones and hymenialdisine (Cohen, P. et al., Nat Rev Drug Discov. 2004, 3, 479-487; Martinez, A., Med Res Rev. 2008, 28, 773-796). Recently, two disclosed GSK3 inhibitors have entered clinical development as disease-modifying drugs for the treatment of Alzheimer's disease. However, kinase selectivity of the presently known inhibitors seems limited and might be critical for further pharmaceutical development. Additionally, all the GSK3 inhibitors developed until now are inhibiting the two isoforms of GSK3, GSK3alpha and beta, with similar potency (Martinez A., Med Res Rev. 2008, 28, 773-796). Thus, the need for further improved inhibitors of GSK3 is strongly indicated.
The present invention provides novel small molecule inhibitors of cyclin-dependent kinases such as CDK9 and/or glycogen synthase kinase 3 family members such as GSK3-alpha and -beta. Suitably, said small molecule inhibitors show selectivity in inhibiting a particular CDK, in particular CDK9, and/or glycogen synthase kinase 3 family members. Said small molecule inhibitors may have a therapeutic utility for the treatment of conditions such as proliferative, immunological, neurodegenerative, infectious and cardiovascular diseases. Furthermore, the small molecule inhibitors of the present invention have surprisingly been shown to exert a beneficial effect in the treatment of inflammatory diseases and of any type of pain.
Current treatments for inflammatory diseases and any type of pain are only partially effective, and many also cause debilitating or dangerous side effects. For example, many of the traditional analgesics used to treat severe pain induce debilitating side effects such as nausea, dizziness, constipation, respiratory depression, and cognitive dysfunction (Brower, V., Nat Biotechnol. 2000, 18, 387-391).
Current approaches for the treatment of inflammation and especially inflammatory pain aim at cytokine inhibition (e.g. IL113) and suppression of pro-inflammatory TNFα. Current approved anticytokine/antiTNFα treatments are based on chimeric antibodies such as Infliximab and Etanercept which reduce TNFα circulation in the bloodstream. TNFα is one of the most important inflammatory mediators inducing synthesis of important enzymes such as COX-2, MMP, iNOS, cPLa2 and others. The main drawbacks of these “biologicals”, however, reside in their immunogenic potential with attendant loss of efficacy and their kinetics that lead to a more or less digital all-or-nothing reduction of circulating TNFα. The latter can result in severe immune suppressive side effects.
Thus, the usual outcome of such treatment is partial or unsatisfactory, and in some cases the adverse effects of these drugs outweigh their clinical usefulness.
In conclusion, there is a high unmet need for safe and effective methods of treatment of inflammatory diseases and pain treatment, in particular of chronic inflammatory and neuropathic pain.
New approaches like fragment-based screening techniques and structure based drug design or knowledge based prediction of ligand binding modes were described in the literature recently (Wyatt, P. G. et al., J Med Chem 2008, 51, 4986-4999; Ghose, A. K. et al., J Med Chem 2008, 51, 5149-5171). A further insight into structural features of CDK9 was performed by the publication of the solved X-ray structure of the complex CDK9/cyclin Ti with flavopiridol (Baumli, S. et al., EMBO J. 2008, 27, 1907-1918).